PML-RARA can increase hematopoietic self-renewal without causing a myeloproliferative disease in mice.

1Section of Stem Cell Biology, Division of Oncology, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63119, USA.

Abstract

Acute promyelocytic leukemia (APL) is characterized by the t(15;17) translocation that generates the fusion protein promyelocytic leukemia-retinoic acid receptor α (PML-RARA) in nearly all cases. Multiple prior mouse models of APL constitutively express PML-RARA from a variety of non-Pml loci. Typically, all animals develop a myeloproliferative disease, followed by leukemia in a subset of animals after a long latent period. In contrast, human APL is not associated with an antecedent stage of myeloproliferation. To address this discrepancy, we have generated a system whereby PML-RARA expression is somatically acquired from the mouse Pml locus in the context of Pml haploinsufficiency. We found that physiologic PML-RARA expression was sufficient to direct a hematopoietic progenitor self-renewal program in vitro and in vivo. However, this expansion was not associated with evidence of myeloproliferation, more accurately reflecting the clinical presentation of human APL. Thus, at physiologic doses, PML-RARA primarily acts to increase hematopoietic progenitor self-renewal, expanding a population of cells that are susceptible to acquiring secondary mutations that cause progression to leukemia. This mouse model provides a platform for more accurately dissecting the early events in APL pathogenesis.

(A) Schema showing that mice were treated with 10 doses of tamoxifen (4 mg i.p. twice weekly for 5 weeks) and analyzed 2 weeks later. (B) mPML-fPR modestly decreased the percentage of Lin–cKit+Sca+ (KLS) cells but not the percentage of Lin–cKit+Sca–CD16/32+CD34+ (GMP), Lin–cKit+Sca–CD16/32–CD34+ (CMP), or Lin–cKit+Sca–CD16/32–CD34– (MEP) cells. KLS is expressed as the percentage of Lin– cells; the others are expressed as the percentage of Lin–cKit+Sca– cells. Results are from 4 independent mice in each genotype. Horizontal bars are the median value. (C) mPML-fPR did not alter the number of CFUs in the bone marrow cells when grown for 7 days in methylcellulose containing IL-3, IL-6, and SCF (average ± SD). (D) mPML-fPR did not alter the immunophenotype of cells maturing during the methylcellulose culture in C. After 1 week in culture, cells were collected from methylcellulose, washed, and stained as indicated. Representative results are from 2 experiments (average ± SD). Horizontal bars are the median value. (E) Acute exposure of ERT2-Cre+/– × mPML-PRflox+/– bone marrow cells to 4-hydroxytamoxifen (4-OH tam) (0.1 M or 1 M) modestly decreased the number of CFU-Ms and total CFUs. Bone marrow cells from mice without prior tamoxifen exposure were harvested and plated in methylcellulose, as above, containing increasing doses of 4-hydroxytamoxifen (average ± SD).

(A) Experiment schema showing that ERT2-Cre+/– × mPML-PRflox+/– mice and littermate controls were treated with tamoxifen (4 mg i.p. twice weekly for 10 doses). Bone marrow cells were plated in methylcellulose containing IL-3, IL-6, and SCF. At 7-day intervals, colony numbers were counted and cells were harvested and replated as indicated. (B) mPML-PRflox leads to inappropriate self-renewal when intercrossed with ERT2-Cre but not LysM-Cre. Representative results are from 2 experiments. Data points represent average and SD from 4 individual mice. (C) The mPML-fPR allele was associated with continued CFU activity in ERT2-Cre, but not LysM-Cre, bone marrow. The percentage of mPML-fPR alleles was assessed at each time point in B using qPCR. Data points represent results from individual mice. Horizontal bars are the median value.